The complexity of intracellular signaling is demonstrated in the crosstalk between various signaling pathways and in their regulation by the ubiquitous analytes, such as ATP, ROS and cell metabolites, with mitochondria at the center stage. Thus, it is critical to monitor real-time activities of mitochondria to fully understand ther impact on cell signaling. Here, this team proposes to investigate if and how mitochondrial fission/fusion events, collectively called mtDynamics, modulate signaling events during environmental carcinogenesis by regulating mitochondrial ROS production in real time. To investigate such a precise role of mitochondria, we propose to develop a quantitative microscopy-based analytical method of Real Time Simultaneous Quantitative Assessment of the Redox Environment and Dynamics of Mitochondria (RT- SQuARED-M). In the R21 phase the performance of the RT-SQuARED-M method will be further developed and validated. After meeting the set R21 milestone, a broader scientific hypothesis in the R33 phase will be tested. The pioneering studies and new preliminary data demonstrate that repression of mtFission, which allows for unopposed mtFusion and repressed mitophagy, leads to a) increased cell proliferation, b) enhanced mtROS production from hotspots, and c) non-canonical recruitment of crucial cell cycle regulators on mitochondria. Based on these data, it is hypothesized that carcinogenicity caused by oxidative damage-inducing environmental polyhydrocarbons is initiated as mtDynamics-driven local mtROS modulates distinct mitochondrial sites for signaling and mitophagy. Using the RT-SQuARED-M method, we anticipate discovering 1) lower mtROS at the DRP1-driven mtFission sites define the mitophagic hotspots, while the higher mtROS at the MFN1/2-driven mtFusion sites define the signaling hotspots; 2) polyhydrocarbon carcinogens B[a]P and TCDD lower Drp1 driven-mtFission to repress mitophagic clearance and allow aberrant mtROS-based growth factor-cell cycle signaling in mitochondria to initiate lung and skin carcinogenesis. Discovery of such precise mitochondrial signaling that could be modulated by environmental carcinogens would be a paradigm shift in the role of mitochondria in environmental toxicity. This study, if successful, will reveal the mechanism of action behind the involvement of mitochondrial dynamics in lung and skin carcinogenesis and highlight key mitochondrial fission/fusion proteins (Drp1 and/or Mfn1/2) as potential targets for preventing initiation of lung and skin carcinogenesis. The proposed research team has expertise in all the methods and procedures required for successful development and application of the innovative RT- SQuARED-M method in understanding carcinogenesis of the lung and skin.